FIG 2 .

The Opal524R mutation alters levels of nsP34 and mature nsP4 within infected cells. (A, B) Vero cells were infected in duplicate at a multiplicity of infection (MOI) of 5 with wild-type CHIKV or the Opal524R mutant. (A) At the indicated times postinfection (hours postinfection [hpi]), protein was extracted for Western blotting. Blots were probed for the indicated markers. M, mock. (B) Densitometry was performed for nsP3 and E2 using ImageJ software. Band intensity was normalized against that of actin, and the averages of intensities in duplicate lanes from a representative blot are shown. Data are representative of two independent experiments. (C, D) CHIKV replicons containing either the wild-type sequence or the Opal524R mutation were generated. Each replicon contains a C-terminal 3× FLAG tag on nsP4 (nsP4-FLAG). BHK-21 cells were electroporated with 10 μg of Sp6-transcribed RNA of the indicated replicon or mock electroporated in 1× PBS. (C) At the indicated times postelectroporation (hours postelectroporation [hpe]), protein was extracted for Western blotting. (D) Densitometry was performed for nsP3, nsP4-FLAG, and nsP34-FLAG using ImageJ software. Band intensity was normalized against that of actin. Data are representative of two independent experiments.